Misuse of controlled substances, including drugs of abuse and steroids, have recently become the focus of media attention and regulatory activity in both the private and public sectors. Many agreements between athletes and the management of professional sports teams provide for random testing for drugs of abuse such as opiates, cocaine and cannabis.
Likewise, there are now provisions which require drug testing for many federal and state employees in key positions. Moreover, emergency situations due to drug overdose and poisoning require immediate diagnosis and treatment. Often, in such emergency situations the results of blood, urine and saliva tests which must be sent to laboratories, arrive too late to save the victim. Many of these tests are difficult to administer and require trained personnel who are often unavailable in emergency situations. Accordingly, there has long been a need for an assay which is simple to administer and which provides an immediate and accurate determination of the presence of a specific drug or poison in the body fluids of the test subject, whether the subject is an incoherent or unconscious victim, or a suspected drug abuser.
A variety of immunoassays have previously been developed, these include: radioimmunoassays ("RIA"), homogeneous enzyme-multiplied immunoassays ("EMIT"), enzyme linked immunoadsorbent assays ("ELISA"), apoenzyme reactivation immunoassay ("ARIS"), dipstick immunoassays and immunochromotography assays.
The most widely used of these are the RIA; see, Nabarro, Radioimmunoassay and Saturation Analysis, Brit. Med. Bull, 30, 1-103 (1974); the ELISA, see, Voller, et al., The Enzyme Linked Immunosorbent Assay (ELISA). Dynatech Laboratories, Inc., Alexandria, Va. 1-125 (1979); and the EMIT, see, Rubenstein, et al., "Homogeneous" Enzyme Immunoassay. A New Immunochemical Technique, BBRC, 47, 846-851 (1972).
RIA techniques are very sensitive, however, the radioactive isotopes used in these techniques require complex equipment for reading the results of the RIA and special safety measures for the use and disposal of the radioactive isotopes. Heterogeneous enzyme immunoassays such as the ELISA, require the separation of enzyme-labeled substance from unreacted enzyme-labeled material. These assays are most commonly used for the analysis of large molecules.
An improvement to the heterogeneous binding immunoassays is described by Cais, in U.S. Pat. No. 4,205,952 for the detection of various metabaloids, drugs and the like. The technique utilizes a binding component, and a labelled metallic constituent that can be detected. The labelled metallic constituent includes one or more metal atoms which can be detected, and are present in the form of a variety of metalo organic derivaties or metal coordination complexes. Although this technique does not use radioactive isotopes, it is time consuming, complex and still requires a variety of equipment for the separation of the bound-phase from the free-phase by filtration or centrifugation, and the detection of metals by emission, absorption, and fluorescence spectronomy.
In the detection of small molecules, however, the EMIT system is most prevalent. In the EMIT system, a hapten is linked to an enzyme and the activity of the enzyme is inhibited when an antibody is combined with the hapten-enzyme conjugate. The assay system contains an enzyme-hapten also called an "enzyme-analyte", an antibody, an enzyme substrate and a test sample. If the test sample contains any of the analyte, it will combine with the antibody leaving the enzyme-analyte conjugate free to react with the substrate. Conversely, if there is no analyte present in the test sample, the activity of the enzyme will be inhibited by its interaction with the antibody.
Refinements in the EMIT techniques have resulted in more rapid and accurate immunoassays. For example, Karmen et al., U.S. Pat. No. 4,600,690 disclose an EMIT type immunoassay. In this immunoassay an excess amount of unlabeled antigen is added after the start of the reaction between the labeled hapten (ligand), the unlabeled ligand (hapten) and the antibody, in order to saturate the binding sides of the antibody. This technique provides increased sensitivity in the competitive binding immunoassay. However, it requires precise measurements in the laboratory and highly trained personnel to conduct the assay. In addition, it does not provide for instantaneous test results.
Recently, attention has focused on placing the components of these immunoassays on a solid phase. The goal is to produce a one step dipstick immunoassay. A comprehensive discussion of the use of reagent strips for various immunoassays has been co-authored by the inventor, herein, and is will soon be published. See, Emancipator, K., and Deutsch, D. G., Dry Reagent Chemistries in Toxicology, (Ed. Deutsch, D. G.), in Analytical Aspects of Clinical Toxicology, Chemical Analysis Series, Wiley, N.Y. (In Press 1988). For example, as discussed therein, Litman, et al. disclose a qualitative dipstick for testing of morphine in urine; see, Litman, et al., An Internally Referenced Test Strip Immunoassay for Morphine, Clin. Chem. 29, 1958-1603 (1983). This immunoassay uses the enzymechanneling of glucose oxidase-horseradish peroxidase to immunospecifically generate an insoluble reaction product on the surface of a test strip. The test strip also contains an antibody specific for morphine which inhibits the development of color on the test strip when the antibody combines with morphine in the test sample.
There have also been publications describing dry-reagent strips for testing levels of theophylline, a potent bronchodilator used to treat acute and chronic asthmatic symptoms. Concentrations of theophylline over 10 to 20 mg/1 are associated with toxic symptoms, such as nausea, vomiting, headaches, and in extreme cases convulsions and death. A method developed by Rupchock et al., in Dry-Reagent Strips Used for the Determination of Theophylline in Serum, Clin. Chem. 31, 731-740 (1985), which is based in Serum, Clin. Chem. 31, 731-740 (198 upon the ARIS system. Rupchock et al. disclose that theophylline competes with theophylline conjugate labeled with flavine adenine dinucleotide for a limited number of antibody binding sites. The antibody is incorporated into a reagent strip. In the absence of theophylline in the blood serum of the patient, the conjugate binds to the antibody and is not available for further reaction with apoglucose oxidase. Apoglucose oxidase is detected by the presence of a colored product, produced by a coupled reaction of Apoglucose oxidase with peroxidase and other reagents impregnated on the filter paper test strip. The intensity of color is proportional to the concentration of the theophylline in the specimen and can be quantitatively analyzed using a reflectance photometer. One drawback of this method is that it requires the separation of blood serum from whole blood, prior to testing.
Another method which utilizes a solid phase for the detection and analysis of theophylline uses an immunochromatographic assay developed by Zuk et al., in Enzyme Immunochromatography-A Quantitative Immunoassay Requiring No Instrumentation, Clin. Chem., 31, 1144 (1985). This method does not require separation steps and can be performed on whole blood without using any detection instruments. In this method, the sample drug ("antigen") and an antigen-enzyme conjugate are first combined, and a paper strip on which an antibody has been immobilized is dipped into this combined sample, allowing the sample to move up paper strip by capillary action. After color development due to peroxidase reaction, the result of the assay is obtained by reading the height of the colored zone on the test strip. The reaction takes approximately fifteen minutes.
All of the immunoassays which take advantage of the affinity of the hapten-enzyme complex for the antibody in competition with the affinity of the antigen/analyte for the antibody, are subject to specific binding strengths of the enzyme/substrate combination, which can not be varied. It would be desirable, however, for an investigator to have the flexibility to vary the binding strengths of the reagents in an immunoassay, instead of being locked into the classical enzyme-substrate interactions. In addition, the use of enzymes in an immunoassay result in several other disadvantages, including lack of stability due to denaturation of the enzymes, temperature dependance, pH dependance and ionic strength (salt) dependance. For example, many EMIT tests can be fooled into registering a false negative by high salt concentrations or pH variations in the test sample.
Prior to the present invention, nucleic acids have not been used as reagents for immunoassays. Nucleic acids have been used, however, for diagnostic tests, as probes detecting the DNA of infective agents such as that of a virus, or malignant tissue. Thus, Schafritz U.S. Pat. No. 4,562,159 discloses a hybridization probe prepared from hepatitis B-virus DNA (HBV-DNA) labeled with highly radioactive substances such as .sup.32 P or .sup.125 I. The hybridization probe is combined with a test sample suspected of containing hepatitis B-virus. Following incubation, the combined HBV-DNA probe is removed from the substrate and the presence of hybridized HBV-DNA is determined by liquid scintillation spectroscopy or by autoradiography of the substrate. In addition, Gottlieb U.S. Pat. No. 4,490,472 discloses a test for malignancies based on DNA detection. According to this test, sera from a test subject is mixed with labeled DNA in the presence of an enzyme-conjugated resin. Sera from normal and malignant tissue have different degrees of affinity and react differently with the resin, permitting a diagnosis of a malignancy.
Accordingly, an object of the present invention is to provide an immunoassay for testing body fluids for poisons, toxins and drugs of abuse which would permit quick screening in emergency situations.
Another object of the present invention is to provide an assay which allows personnel which are not familiar with complex laboratory techniques to use the assays in emergency situations.
A further object of the present invention is to provide an assay which allows for the custom preparation of reagents with specific binding strengths to provide an extremely reliable and sensitive immunoassay.
Still, a further object of the present invention is to provide an immunoassay with reagents which are extremely stable and relatively insensitive to variations in temperature, pH and ionic concentrations of the sample.